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Proc Natl Acad Sci U S A. 2016 Oct 18;113(42):11853-11858. Epub 2016 Oct 4.

Real-time analysis of RAG complex activity in V(D)J recombination.

Author information

1
Department of Biochemistry and Molecular Pharmacology, Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016.
2
Department of Pathology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Biochemistry & Molecular Biology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Molecular Microbiology & Immunology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Section of Molecular & Computational Biology, Department of Biological Sciences, University of Southern California (USC), Los Angeles, CA 90089.
3
Department of Pathology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Biochemistry & Molecular Biology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Molecular Microbiology & Immunology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Section of Molecular & Computational Biology, Department of Biological Sciences, University of Southern California (USC), Los Angeles, CA 90089 eli.rothenberg@nyumc.org lieber@usc.edu.
4
Department of Biochemistry and Molecular Pharmacology, Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016; eli.rothenberg@nyumc.org lieber@usc.edu.

Abstract

Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS-RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

KEYWORDS:

V(D)J recombination; antigen receptor gene; immunoglobulin; recombination-activating gene; single-molecule FRET

PMID:
27702897
PMCID:
PMC5081608
DOI:
10.1073/pnas.1606721113
[Indexed for MEDLINE]
Free PMC Article

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